Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein.
The three-dimensional crystal structure of certain DNA polymerases has revealed three separate subdomains, named palm, fingers and thumb (Joyce, C. M. and Steiz, T. A. (1994) Function and structure relationships in DNA polymerases, Annu. Rev. Biochem., 63, 777-822), each having key roles during DNA polymerisation.
The C terminal thumb subdomain of DNA polymerases has been implicated in DNA binding and processivity (Doublie et al. 1998. Nature 391, 251; Truniger et al. 2004. Nucleic Acids Research 32, 371). Residues in this region of DNA polymerases interact with the primer:template duplex.
Disruption of the structure of this region either by the introduction of site-directed mutations or truncation by the deletion of a small number of amino acids, has provided evidence for variants with reduced DNA affinity and processivity without gross changes in other physical properties such as dNTP affinity and nucleotide insertion fidelity (Truniger et al. 2004. Nucleic Acids Research 32, 371; Minnick et al. 1996. J. Biol. Chem., 271, 24954; Polesky et al. 1990. J. Biol. Chem., 265, 14579).
Polymerases may be separated into two structurally distinct families called family A and family B.
The C-terminal subdomain of family B polymerases has been poorly studied, but is believed to be involved in DNA binding based primarily on the inspection of the x-ray crystal structure of the closed form (DNA-bound) of polymerase RB69. Mutagenesis studies have been conducted within this thumb domain for two examples of the family B class, namely Phi29 and T4. However, these studies were limited to amino acid deletions of large portions of the domain. The same type of deletion has been carried out for Klenow (a family A polymerase). The performance of the variants in these studies was evaluated in terms of their ability to bind and incorporate dNTPs, the effect the deletion had on fidelity, their affinity for DNA and also their interaction with accessory proteins.
No studies of the thumb domain of the polymerase from a thermophilic archaeon have previously been carried out.
The subject matter of the present invention was presented in prior filed U.K. Provisional Application No. 0509508.8 filed May 10, 2005, priority of which is believed to be available under 35 U.S.C. § 119, and the disclosure of which is incorporated herein in its entirety. In said application, the structural aspects of the polymerases and the related materials of the present invention were disclosed as they are herein, and were accompanied by information providing further background and characterization of function, which was also in accordance with the understanding of the inventors at the time. Since filing said application, further study of the enzymes in question has taken place and additional data illustrating and advancing the understanding of their function and application, has resulted, which is now felt to be desirably presented herein.
Accordingly, it is toward the advancement of the understanding and application of the present invention that the present application is directed.